Methods for making Apo-2 ligand using divalent metal ions

ABSTRACT

Methods of making Apo-2 ligand and formulations of Apo-2 ligand using divalent metal ions are provided. Such divalent metal ions include zinc and cobalt which improve Apo-2 ligand trimer formation and stability. The crystal structure of Apo-2 ligand is also provided, along with Apo-2 ligand variant polypeptides identified using oligonucleotide-directed mutagenesis.

RELATED APPLICATIONS

This is a continuation application claiming priority to U.S. applicationSer. No. 09/603,866 filed Jun. 26, 2000, now abandoned, which is anon-provisional application claiming priority under Section 119(e) toprovisional application No. 60/141,342 filed Jun. 28, 1999, the contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to making Apo-2 ligand and Apo-2ligand formulations using divalent metal ions, such as zinc or cobalt.The use of such Apo-2 ligand and Apo-2 ligand formulations havingimproved Apo-2L trimer formation and stability is also provided. Thepresent invention also relates to Apo-2 ligand variants, particularlyalanine substitution variants.

BACKGROUND OF THE INVENTION

Control of cell numbers in mammals is believed to be determined, inpart, by a balance between cell proliferation and cell death. One formof cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al.,Bio/Technology, 12:487-493 (1994); Steller et al., Science,267:1445-1449 (1995)]. Apoptotic cell death naturally occurs in manyphysiological processes, including embryonic development and clonalselection in the immune system [Itoh et al., Cell, 66:233-243 (1991)].

Various molecules, such as tumor necrosis factor-α (“TNF-α”), tumornecrosis factor-β (“TNF-β” or “lymphotoxin-α”), lymphotoxin-β (“LT-β”),CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1ligand (also referred to as Fas ligand or CD95 ligand), Apo-2 ligand(also referred to as TRAIL, AIM-1 or AGP-1), and Apo-3 ligand (alsoreferred to as TWEAK) have been identified as members of the tumornecrosis factor (“TNF”) family of cytokines [See, e.g., Gruss and Dower,Blood, 85:3378-3404 (1995); Pitti et al., J. Biol. Chem.,271:12687-12690 (1996); Wiley et al., Immunity, 3:673-682 (1995);Browning et al., Cell, 72:847-856 (1993); Armitage et al. Nature,357:8.0-82 (1992), WO 97/01633 published Jan. 16, 1997; WO 97/25428published Jul. 17, 1997; WO 97/46686 published Dec. 11, 1997; WO97/33899 published Sep. 18, 1997; Marsters et al., Curr. Biol.,8:525-528 (1998); Chicheportiche et al., Biol. Chem., 272:32401-32410(1997)]. Among these molecules, TNF-α, TNF-β, CD30 ligand, 4-1BB ligand,Apo-1 ligand, Apo-2 ligand (TRAIL) and Apo-3 ligand (TWEAK) have beenreported to be involved in apoptotic cell death.

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1)and 75-kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem.,264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci.,87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991] and human andmouse cDNAs corresponding to both receptor types have been isolated andcharacterized [Loetscher et al., Cell, 61:351 (1990); Schall et al.,Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewiset al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al.,Mol. Cell. Biol., 11:3020-3026 (1991)]. Extensive polymorphisms havebeen associated with both TNF receptor genes [see, e.g., Takao et al.,Immunogenetics, 37:199-203 (1993)]. Both TNFRs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors are found naturally also as soluble TNF-binding proteins[Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A., 87:8331 (1990)]. The cloning of recombinantsoluble TNF receptors was reported by Hale et al. [J. Cell. Biochem.Supplement 15F, 1991, p. 113 (P424)].

The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2)contains a repetitive amino acid sequence pattern of four cysteine-richdomains (CRDs) designated 1 through 4, starting from the NH₂-terminus.Each CRD is about 40 amino acids long and contains 4 to 6 cysteineresidues at positions which are well conserved [Schall et al., supra;Loetscher et al., supra; Smith et al., supra; Nophar et al., supra;Kohno et al., supra]. In TNFR1, the approximate boundaries of the fourCRDs are as follows: CRD1—amino acids 14 to about 53; CRD2—amino acidsfrom about 54 to about 97; CRD3—amino acids from about 98 to about 138;CRD4—amino acids from about 139 to about 167. In TNFR2, CRD1 includesamino acids 17 to about 54; CRD2—amino acids from about 55 to about 97;CRD3—amino acids from about 98 to about 140; and CRD4—amino acids fromabout 141 to about 179 [Banner et al., Cell, 73:431-435 (1993)]. Thepotential role of the CRDs in ligand binding is also described by Banneret al., supra.

A similar repetitive pattern of CRDs exists in several othercell-surface proteins, including the p75 nerve growth factor receptor(NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J.,8:1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J., 9:1063(1990)] and the Fas antigen [Yonehara et al., J. Exp. Med.,169:1747-1756 (1989) and Itoh et al., Cell, 66:233-243 (1991)]. CRDs arealso found in the soluble TNFR (sTNFR)-like T2 proteins of the Shope andmyxoma poxviruses [Upton et al., Virology, 160:20-29 (1987); Smith etal., Biochem. Biophys. Res. Commun., 176:335 (1991); Upton et al.,Virology, 184:370 (1991)]. Optimal alignment of these sequencesindicates that the positions of the cysteine residues are wellconserved. These receptors are sometimes collectively referred to asmembers of the TNF/NGF receptor superfamily. Recent studies on p75NGFRshowed that the deletion of CRD1 [Welcher, A. A. et al., Proc. Natl.Acad. Sci. USA, 88:159-163 (1991)] or a 5-amino acid insertion in thisdomain [Yan, H. and Chao, M. V., J. Biol. Chem., 266:12099-12104 (1991)]had little or no effect on NGF binding [Yan, H. and Chao, M. V., supra].p75 NGFR contains a proline-rich stretch of about 60 amino acids,between its CRD4 and transmembrane region, which is not involved in NGFbinding [Peetre, C. et al., Eur. J. Hematol., 41:414-419 (1988);Seckinger, P. et al., J. Biol. Chem., 264:11966-11973 (1989); Yan, H.and Chao, M. V., supra]. A similar proline-rich region is found in TNFR2but not in TNFR1.

The TNF family ligands identified to date, with the exception oflymphotoxin-α, are type II transmembrane proteins, whose C-terminus isextracellular. In contrast, most receptors in the TNF receptor (TNFR)family identified to date are type I transmembrane proteins. In both theTNF ligand and receptor families, however, homology identified betweenfamily members has been found mainly in the extracellular domain(“ECD”). Several of the TNF family cytokines, including TNF-α, Apo-1ligand and CD40 ligand, are cleaved proteolytically at the cell surface;the resulting protein in each case typically forms a homotrimericmolecule that functions as a soluble cytokine. TNF receptor familyproteins are also usually cleaved proteolytically to release solublereceptor ECDs that can function as inhibitors of the cognate cytokines.

Recently, other members of the TNFR family have been identified. Suchnewly identified members of the TNFR family include CAR1, HVEM andosteoprotegerin (OPG) [Brojatsch et al., Cell, 87:845-855 (1996);Montgomery et al., Cell, 87:427-436 (1996); Marsters et al., J. Biol.Chem., 272:14029-14032 (1997); Simonet et al., Cell, 89:309-319 (1997)].Unlike other known TNFR-like molecules, Simonet et al., supra, reportthat OPG contains no hydrophobic transmembrane-spanning sequence. OPG isbelieved to act as a decoy receptor, as discussed below.

Another new member of the TNF/NGF receptor family has been identified inmouse, a receptor referred to as GITR for “glucocorticoid-induced tumornecrosis factor receptor family-related gene” [Nocentini et al., Proc.Natl. Acad. Sci. USA 94:6216-6221 (1997)]. The mouse GITR receptor is a228 amino acid type I transmembrane protein that is expressed in normalmouse T lymphocytes from thymus, spleen and lymph nodes. Expression ofthe mouse GITR receptor was induced in T lymphocytes upon activationwith anti-CD3 antibodies, Con A or phorbol 12-myristate 13-acetate.

In Marsters et al., Curr. Biol., 6:750 (1996), investigators describe afull length native sequence human polypeptide, called Apo-3, whichexhibits similarity to the TNFR family in its extracellularcysteine-rich repeats and resembles TNFR1 and CD95 in that it contains acytoplasmic death domain sequence [see also Marsters et al., Curr.Biol., 6:1669 (1996)]. Apo-3 has also been referred to by otherinvestigators as DR3, wsl-1, TRAMP, and LARD [Chinnaiyan et al.,Science, 274:990 (1996); Kitson et al., Nature, 384:372 (19961; Bodmeret al., Immunity, 6:79 (1997); Screaton et al., Proc. Natl. Acad. Sci.,94:4615-4619 (1997)].

Pan et al. have disclosed another TNF receptor family member referred toas “DR4” [Pan et al., Science, 276:111-113 (1997)]. The DR4 was reportedto contain a cytoplasmic death domain capable of engaging the cellsuicide apparatus. Pan et al. disclose that DR4 is believed to be areceptor for the ligand known as Apo-2 ligand or TRAIL.

In Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science,277:815-818 (1997), another molecule believed to be a receptor for theApo-2 ligand (TRAIL) is described. That molecule is referred to as DR5(it has also been alternatively referred to as Apo-2; TRAIL-R2, TRICK2or KILLER [Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak etal., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics,17:141-143 (1997)]. Like DR4, DR5 is reported to contain a cytoplasmicdeath domain and be capable of signaling apoptosis.

Yet another death domain-containing receptor, DR6, was recentlyidentified [Pan et al., FEBS Letters, 431:351-356 (1998)]. Aside fromcontaining four putative extracellular domains and a cytoplasmic death,domain, DR6 is believed to contain a putative leucine-zipper sequencethat overlaps with a proline-rich motif in the cytoplasmic region. Theproline-rich motif resembles sequences that bind to src-homology-3domains, which are found in many intracellular signal-transducingmolecules.

A further group of recently identified TNFR family members are referredto as “decoy receptors,” which are believed to function as inhibitors,rather than transducers of signaling. This group includes DCR1 (alsoreferred to as TRID, LIT or TRAIL-R3) [Pan et al., Science, 276:111-113(1997); Sheridan et al., Science, 277:818-821 (1997); McFarlane et al.,J. Biol. Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters,416:329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170(1997); and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)] and DCR2(also called TRUNDD or TRAIL-R4) [Marsters et al., Curr. Biol.,7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998);Degli-Esposti et al., Immunity, 7:813-820 (1997)], both cell surfacemolecules, as well as OPG [Simonet et al., supra] and DCR3 [Pitti etal., Nature, 396:699-703 (1998)], both of which are secreted, solubleproteins.

For a review of the TNF family of cytokines and their receptors, seeAshkenazi et al., Science, 281:1305-1308 (1998); Golstein, Curr. Biol.,7:750-753 (1997); and Gruss and Dower, supra.

While zinc binding sites have been shown to play structural roles inprotein-protein interactions in certain proteins involving diverseinterfaces [Feese et al., Proc. Natl. Acad. Sci., 91:3544-3548 (1994);Somers et al., Nature, 372:478-481 (1994); Raman et al., Cell,95:939-950 (1998)], none of the previously structurally-characterizedmembers of the TNF family (CD40 ligand, TNF-alpha, or TNF-beta) bindmetals. The use of metal ions, such as zinc, in formulations of varioushormones, such as human growth hormone (hGH), has been described in theliterature. [See, e.g., WO 92/17200 published Oct. 15, 1992). Zincinvolvement in hGH binding to receptors was likewise described in WO92/03478 Published Mar. 5, 1992. The roles of zinc binding ininterferon-alpha dimers and interferon-beta dimers were reported inWalter et al., Structure, 4:1453-1463 (1996) and Karpusas et al., Proc.Natl. Acad. Sci., 94:11813-11818 (1997), respectively.

The structures and biological roles of various metal ions such as zinchave been reviewed in the art, see, e.g., Christianson et al., Advancesin Protein Chemistry, 42:281-355 (1991).

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the inclusion ofone or more divalent metal ions in methods or processes for making Apo-2ligand, or formulations containing Apo-2 ligand, results in increasedyield and stability of Apo-2 ligand trimers. It is presently believedthat such inclusion of one or more divalent metal ions may also improvefolding of Apo-2 ligand or Apo-2L trimer assembly upon expression inrecombinant cell culture. In oxidative environments, free cysteines onApo-2L monomers may form intermolecular disulfide bridges, giving riseto free-standing Apo-2L dimers as well as disulfide-linked Apo-2L dimerspecies within trimeric forms of Apo-2L. Such formation of Apo-2L dimersmay lead to aggregation, precipitation, and/or inactivation of Apo-2L.The presence of divalent metal ions in the methods and formulationsdescribed herein may protect against such disulfide bond formation. Itappears that inclusion of divalent metal ions during the process ofsynthesis and assembly of Apo-2L trimers may further improveaccumulation and recovery of properly folded, homotrimeric Apo-2L.Applicants have found that Apo-2 ligand trimers are approximately10-fold more active (in cytotoxic activity in mammalian cancer cells) ascompared to disulfide-linked Apo-2L dimers.

While the description of the invention herein is primarily directed toApo-2 ligand, the use of divalent metal ions to make or stabilizetrimers of various other proteins is contemplated. Such other proteinsparticularly include those proteins which require trimer formation forbiological activity, for instance, various members of the TNF family.

In one embodiment, the invention provides a method of making Apo-2ligand using one or more divalent ions. The methods include the steps ofproviding a host cell comprising a replicable vector containing anucleic acid encoding Apo-2 ligand, providing culture media containingone or more divalent metal ions, culturing the host cell in the culturemedia under conditions sufficient to express the Apo-2 ligand, andrecovering the Apo-2 ligand from the host cells or the cell culturemedia. Optionally, one or more divalent metal ions are used during therecovery or purification process.

In another embodiment, the invention provides a formulation comprisingApo-2 ligand and one or more divalent metal ions. The composition may bea pharmaceutically acceptable formulation useful, for instance, ininducing or stimulating apoptosis in mammalian cancer cells.

A further embodiment of the invention provides articles of manufactureand kits that include such Apo-2 ligand formulations containing one ormore divalent metal ions. The articles of manufacture and kits include acontainer, a label on the container, and a formulation contained withinthe container. The label on the container indicates that the formulationcan be used for certain therapeutic or non-therapeutic applications. Theformulation contains Apo-2 ligand and one or more divalent ions.

In another embodiment, the invention provides Apo-2 ligand polypeptidesmade in accordance with the methods described herein. Such Apo-2 ligandsmay comprise amino acids 114-281 of FIG. 1 (SEQ ID NO:1), amino acids1-281 of FIG. 1 (SEQ ID NO:1), as well as biologically active fragmentsor variants thereof.

In a still further embodiment, the invention provides Apo-2 ligandvariants. Particularly, the invention provides Apo-2 ligand variantscomprising one or more amino acid substitutions in the native sequenceof Apo-2 ligand (FIG. 1; SEQ ID NO:1). Apo-2 ligand variants comprisingalanine substitutions are provided in Table I below. The invention alsoprovides nucleic acid molecules encoding such Apo-2L variants andvectors and host cells containing nucleic acid molecules encoding theApo-2L variants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of human Apo-2 ligand cDNA (SEQ IDNO:2) and its derived amino acid sequence (SEQ ID NO:1). The “N” atnucleotide position 447 (in SEQ ID NO:2) is used to indicate thenucleotide base may be a “T” or “G”.

FIG. 2 provides the crystal structure of Apo-2L. FIG. 2A shows a view ofthe trimer along the three fold axis. Each monomer is identical. Theordered protein structure commences at residue 120, residues 131-141 aredisordered, as are residues 195-201 (marked as dashed lines). The zincbinding site including the three symmetry related cysteines and thesolvent ligand are shown as space filling diagrams. FIG. 2B providescross-eyed stereo close up view of the zinc binding site; the anglesbetween Sγ-zinc-Sγ are 112° and the Sγ-zinc-solvent angles are 107° with2.3 Angstrom zinc-sulfur and 2.3 Angstrom zinc-solvent bond distances.FIGS. 2 (and 5) were made with the programs Molscript [Kraulis et al.,J. Appl. Cryst., 24:946-950 (1991)] and Raster3D [Merrit et al., ActaCryst., D50:869-873 (1994)]. FIG. 2C provides a summary of thecrystallographic data from the experiment described in Example 2.

FIG. 3 shows a sequence alignment of selected TNF family members: Apo2L(SEQ ID NO:1); TNF-beta (SEQ ID NO:3); TNF-alpha (SEQ ID NO:4); CD40L(SEQ ID NO:5); FasL (SEQ ID NO:6); RANKL (SEQ ID NO:7). Arrows over thesequence indicate beta-strands in Apo2L. The numbering over the alignedsequences corresponds to the Apo2L sequence numbering provided in FIG. 1(SEQ ID NO:1).

FIG. 4 provides bioassay data showing the functional importance of thezinc binding site. SK-MES-1 cell viability was determined by afluorescence assay of metabolic activity after overnight incubation withvarious concentrations of Apo-2L (form 114-281), or Apo-2L (form114-281) treated with chelating agents to remove the zinc.

FIG. 5 shows mutational analysis mapped onto a space-filling model ofApo-2L. The trimer is oriented as in FIG. 2. Residues with a greaterthan 5-fold decrease in bioactivity when mutated to alanine are labeledand darkly shaded. Other residues that have been mutated are shown inmedium shading and a few of these residues are also labeled.

FIG. 6 shows circular dichroic spectra of Apo-2L (form 114-281) beforeand after treatment to remove the bound zinc.

FIG. 7 shows thermal denaturation of Apo-2L before and after zincremoval monitored by circular dichroism at 225 nm. The dynode voltage isreported for 2 micromolar solutions of Apo-2L.

FIG. 8 shows the effect (time course) of ZnSO₄ additions on solubleApo-2L product accumulation (gm/L) in an E. coli expression system usingan AP promoter.

FIG. 9 shows the elution profiles from MPHS chromatography of celllysates from the E. coli expression system (see Example 8) conducted inthe presence or absence of ZnSO₄.

FIG. 10 shows the effect (time course) of ZnSO₄ addition on solubleApo-2L product accumulation (gm/L) in an E. coli expression system usinga trp promoter.

FIG. 11 shows the effect (time course) of CoCl₂ addition on solubleApo-2L product accumulation (gm/L) in an E. coli expression system usingan AP promoter.

FIG. 12 shows the pAPApo2-P2RU plasmid construct.

FIG. 13 shows the pAPOK5 plasmid construct.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “Apo-2 ligand”, “Apo-2L”, and “TRAIL” are used herein to referto a polypeptide sequence which includes amino acid residues 114-281,inclusive, 95-281, inclusive, residues 92-281, inclusive, residues91-281, inclusive, residues 41-281, inclusive, residues 15-281,inclusive, or residues 1-281, inclusive, of the amino acid sequenceshown in FIG. 1 (SEQ ID NO:1), as well as biologically active fragments,deletional, insertional, or substitutional variants of the abovesequences. In one embodiment, the polypeptide sequence comprisesresidues 114-281 of FIG. 1 (SEQ ID NO:1). Optionally, the polypeptidesequence comprises residues 92-281 or residues 91-281 of FIG. 1 (SEQ IDNO:1). The Apo-2L polypeptides may be encoded by the native nucleotidesequence shown in FIG. 1 (SEQ ID NO:2). Optionally, the codon whichencodes residue Pro119 (FIG. 1; SEQ ID NO:2) may be “CCT” or “CCG”. Inanother preferred embodiment, the fragments or variants are biologicallyactive and have at least about 80% amino acid sequence identity, morepreferably at least about 90% sequence identity, and even morepreferably, at least 95%, 96%, 97%, 98%, or 99% sequence identity withany one of the above sequences. The definition encompassessubstitutional variants of Apo-2 ligand in which at least one of itsnative amino acids are substituted by an alanine residue. Preferredsubstitutional variants include one or more of the residue substitutionsidentified in Table I below. The definition also encompasses a nativesequence Apo-2 ligand isolated from an Apo-2 ligand source or preparedby recombinant or synthetic methods. The Apo-2 ligand of the inventionincludes the polypeptides referred to as Apo-2 ligand or TRAIL disclosedin WO97/01633 published Jan. 16, 1997 and WO97/25428 published Jul. 17,1997. The terms “Apo-2 ligand” or “Apo-2L” are used to refer generallyto forms of the Apo-2 ligand which include monomer, dimer or trimerforms of the polypeptide. All numbering of amino acid residues referredto in the Apo-2L sequence use the numbering according to FIG. 1 (SEQ IDNO:1), unless specifically stated otherwise. For instance, “D203” or“Asp203” refers to the aspartic acid residue at position 203 in thesequence provided in FIG. 1 (SEQ ID NO:1).

The term “Apo-2 ligand extracellular domain” or “Apo-2 ligand ECD”refers to a form of Apo-2 ligand which is essentially free oftransmembrane and cytoplasmic domains. Ordinarily, the ECD will haveless than 1% of such transmembrane and cytoplasmic domains, andpreferably, will have less than 0.5% of such domains.

The term “Apo-2 ligand monomer” or “Apo-2L monomer” refers to a covalentchain of an extracellular domain sequence of Apo-2L.

The term “Apo-2 ligand dimer” or “Apo-2L dimer” refers to two Apo-2Lmonomers joined in a covalent linkage via a disulfide bond. The term asused herein includes free standing Apo-2L dimers and Apo-2L dimers thatare within trimeric forms of Apo-2L (i.e., associated with anotherApo-2L monomer).

The term “Apo-2 ligand trimer” or “Apo-2L trimer” refers to three Apo-2Lmonomers that are non-covalently associated.

“TNF family member” is used in a broad sense to refer to variouspolypeptides that share some similarity to tumor necrosis factor (TNF)with respect to structure or function. Certain structural and functionalcharacteristics associated with the TNF family of polypeptides are knownin the art and described, for example, in the above Background of theInvention. Such polypeptides include but are not limited to thosepolypeptides referred to in the art as TNF-alpha, TNF-beta, CD40 ligand,CD30 ligand, CD27 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (alsoreferred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referredto as TRAIL), Apo-3 ligand (also referred to as TWEAK), APRIL, OPGligand (also referred to as RANK ligand, ODF, or TRANCE), and TALL-1(also referred to as BlyS, BAFF or THANK) [See, e.g., Gruss and Dower,Blood, 85:3378-3404 (1995); Pitti et al., J. Biol. Chem.,271:12687-12690 (1996); Wiley et al., Immunity, 3:673-682 (1995);Browning et al., Cell, 72:847-856 (1993); Armitage et al. Nature,357:80-82 (1992), WO 97/01633 published Jan. 16, 1997; WO 97/25428published Jul. 17, 1997; Marsters et al., Curr. Biol., 8:525-528 (1998);Chicheportiche et al., Biol. Chem., 272:32401-32410 (1997); Hahne etal., J. Exp. Med., 188:1185-1190 (1998); WO98/28426 published Jul. 2,1998; WO98/46751 published Oct. 22, 1998; WO/98/18921 published May 7,1998; Moore et al., Science, 285:260-263 (1999); Shu et al., J.Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med.,189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem.,274:15978-15981 (1999)].

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising Apo-2 ligand, or a portion thereof, fused to a“tag polypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the Apo-2 ligand. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 to about 50 amino acid residues (preferably, betweenabout 10 to about 20 residues).

The term “divalent metal ion” refers to a metal ion having two positivecharges. Examples of divalent metal ions for use in the presentinvention include but are not limited to zinc, cobalt, nickel, cadmium,magnesium, and manganese. Particular forms of such metals that may beemployed include salt forms (e.g., pharmaceutically acceptable saltforms), such as chloride, acetate, carbonate, citrate and sulfate formsof the above mentioned divalent metal ions. A preferred divalent metalion for use in the present invention is zinc, and more preferably, thesalt form, zinc sulfate. Divalent metal ions, as described herein, arepreferably employed in concentrations or amounts (e.g., effectiveamounts) which are sufficient to, for example, (1) enhance storagestability of Apo-2L trimers over a desired period of time, (2) enhanceproduction or yield of Apo-2L trimers in a recombinant cell culture orpurification method, (3) enhance solubility (or reduce aggregation) ofApo-2L trimers, or (4) enhance Apo-2L trimer formation.

“Isolated,” when used to describe the various proteins disclosed herein,means protein that has been identified and separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that would typically interferewith diagnostic or therapeutic uses for the protein, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the protein will be purified (1) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (2) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated protein includesprotein in situ within recombinant cells, since at least one componentof the Apo-2 ligand natural environment will not be present. Ordinarily,however, isolated protein will be prepared by at least one purificationstep.

An “isolated” Apo-2 ligand nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the Apo-2 ligand nucleic acid. An isolated Apo-2ligand nucleic acid molecule is other than in the form or setting inwhich it is found in nature. Isolated Apo-2 ligand nucleic acidmolecules therefore are distinguished from the Apo-2 ligand nucleic acidmolecule as it exists in natural cells. However, an isolated Apo-2ligand nucleic acid molecule includes Apo-2 ligand nucleic acidmolecules contained in cells that ordinarily express Apo-2 ligand where,for example, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

“Percent (%) amino acid sequence identity” with respect to the sequencesidentified herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe Apo-2 ligand sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art can determine appropriate parameters for measuringalignment, including assigning algorithms needed to achieve maximalalignment over the full-length sequences being compared. For purposesherein, percent amino acid identity values can be obtained using thesequence comparison computer program, ALIGN-2, which was authored byGenentech, Inc. and the source code of which has been filed with userdocumentation in the US Copyright Office, Washington, D.C., 20559,registered under the US Copyright Registration No. TXU510087. TheALIGN-2 program is publicly available through Genentech, Inc., South SanFrancisco, Calif. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Biologically active” or “biological activity” for the purposes hereinmeans (a) having the ability to induce or stimulate apoptosis in atleast one type of mammalian cancer cell or virally-infected cell in vivoor ex vivo; (b) capable of raising an antibody, i.e., immunogenic; (c)capable of binding and/or stimulating a receptor for Apo-2L; or (d)retaining the activity of a native or naturally-occurring Apo-2Lpolypeptide.

The terms “apoptosis” and “apoptotic activity” are used in a broad senseand refer to the orderly or controlled form of cell death in mammalsthat is typically accompanied by one or more characteristic cellchanges, including condensation of cytoplasm, loss of plasma membranemicrovilli, segmentation of the nucleus, degradation of chromosomal DNAor loss of mitochondrial function. This activity can be determined andmeasured, for instance, by cell viability assays, FACS analysis or DNAelectrophoresis.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. Moreparticular examples of such cancers include squamous cell carcinoma,small-cell lung cancer, non-small cell lung cancer, glioma,gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer,colorectal cancer, endometrial cancer, kidney cancer, prostate cancer,thyroid cancer, neuroblastoma, pancreatic cancer, glioblastomamultiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma,breast cancer, colon carcinoma, and head and neck cancer.

The terms “treating”, “treatment” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

II. Compositions and Methods of the Invention

A novel cytokine related to the TNF ligand family, the cytokineidentified herein as “Apo-2 ligand” has been described. The predictedmature amino acid sequence of human Apo-2 ligand contains 281 aminoacids, and has a calculated molecular weight of approximately 32.5 kDa.The absence of a signal sequence and the presence of an internalhydrophobic region suggests that Apo-2 ligand is a type II transmembraneprotein. Soluble extracellular domain Apo-2 ligand polypeptides havealso been described. See, e.g., WO97/25428 published Jul. 17, 1997.Apo-2L substitutional variants have further been described. Alaninescanning techniques have been utilized to identify varioussubstitutional variant molecules having biological activity.

Particular substitutional variants of the Apo-2 ligand include those inwhich at least one amino acid is substituted by an alanine residue.These substitutional variants are identified, for example, as “D203A”;“D218A” and “D269A.” This nomenclature is used to identify Apo-2 ligandvariants wherein the aspartic acid residues at positions 203, 218,and/or 269 (using the numbering shown in FIG. 1 SEQ ID NO:1)) aresubstituted by alanine residues. Optionally, the Apo-2L variants maycomprise one or more of the alanine substitutions which are recited inTable I below.

The x-ray crystal structure of the extracellular domain of Apo-2 ligandis now provided in the present invention, and alanine-scanningmutagenesis has been performed to provide the mapping of its receptorcontact regions. The structure obtained for Apo-2 ligand reveals ahomotrimeric protein which contains a novel divalent metal ion (zinc)binding site that coordinates the interaction of the Apo-2 ligand trimermolecule's three subunits.

The x-ray structure of Apo-2L was determined by molecular replacementusing a model of TNF-alpha [Eck et al., J. Biol. Chem., 264:17595-17605(1989)] and refined to 3.9 Angstrom (for the 114-281 residue form) and1.3 Angstrom (for the D218A variant; 91-281 form). Like other members ofthe TNF family, Apo-2L appears to comprise a compact trimer formed ofthree jelly roll monomers which bury approximately 5100 Angstrom² (1700Angstrom² per monomer) to form the globular trimer (See FIG. 2A). Theposition of the core beta-strands was well conserved compared to theother structurally characterized members of the TNF family, TNF-alpha[Eck et al., supra; Jones et al., Nature, 338:225-228 (1989)], TNF-beta[Eck et al., J. Biol. Chem., 267:2119-2122 (1992)], and CD40L [Karpusaset al., Structure, 3:1031-1039 (1995)], with a r.m.s.d. of 0.8 Angstromwhen compared to the core strands of TNF-alpha or TNF-beta. None of theresidues in the Apo-2L trimer interface appear to be absolutelyconserved across the sequences of the all the presently known human TNFfamily members; however, the hydrophobic chemical nature of theseresidues is preserved. (See FIG. 3). The conserved residues in theApo-2L trimer interface cluster near the base (the widest part of thetrimer) and along the three-fold axis. Near the top of the Apo-2L trimerinterface in the vicinity of Cys230, the structures appear to diverge,and the conformation of the 190's and 230's loops are variable in eachstructure.

In contrast to the beta-scaffold core, the structure of the loops andreceptor binding surfaces varies considerably among the TNF familymembers. One difference between the structure of Apo-2 ligand and thestructures of TNF-alpha, TNF-beta, and CD40L is the connections betweenstrands A and A′. In TNF-alpha, TNF-beta, and CD40L, strand A isfollowed by a compact loop. In Apo-2 ligand, a 15-residue insertionlengthens this loop and alters its conformation. The first part of theloop (residues 131 to 141) is disordered while the second part of theloop (residues 142 to 154) crosses the surface of the molecule from onemonomer-monomer interface to the next (see FIG. 2A) with a conformationthat resembles CD40L in its C-terminal portion.

Applicants surprisingly found a novel divalent metal ion (zinc) bindingsite buried near the top of the trimerization interface. The TNF familymembers can be divided by sequence analysis into three groups withrespect to Cys230: (1) proteins such as TNF-alpha and Fas ligand inwhich a cysteine residue at the position corresponding to Cys230 isaccompanied by another cysteine in the adjacent loop (the 194-203 loopin Apo-2L) with which it can form a disulfide bridge precluding it frominteracting with a metal ion, (2) proteins without a cysteinecorresponding to Cys230 (such as TNF-beta and OPGL), and (3) proteinswhich have only one cysteine residue corresponding to Cys230. Apo-2L andits orthologs in other species meet the latter criteria (i.e., proteinswhich have only Cys230) and are expected to bind divalent metal ions atthe trimer surface. The conformation of the main chain immediately priorto Cys230 in Apo-2L differs from the disulfide containing TNF familymembers such as TNF-alpha and CD40L. In Apo-2L, the side chain of Cys230is oriented towards the interface instead of away from it.

The Cys230 residue in each Apo-2L monomer point inward toward the trimeraxis and coordinate a divalent metal ion in conjunction with an interiorsolvent molecule. This divalent metal ion binding site exhibits slightlydistorted tetrahedral geometry with bonds and angles appropriate for azinc binding site and is completely inaccessible to solvent (see FIG.2B). The identity of the bound metal was confirmed using inductivelycoupled plasma atomic emission spectrometry (ICP-AES) (see Example 5).In a quantitative analysis for Cd, Co, Zn, Ni, and Cu using ICP-AES,0.79 moles of Zn and 0.06 moles of Co per molecule of Apo-2L trimer weredetected demonstrating that the bound ion in the structure was zinc atapproximately a one to one molar ratio (see Example 5). The importanceof this site was demonstrated by the observation that alaninesubstitution of Cys230 resulted in a >8-fold decreased apoptoticactivity (See Example 7). Furthermore, removal of the bound metal fromApo-2L by dialysis against chelating agents resulted in a 7-folddecrease in DR5 affinity and a >90-fold decrease in apoptotic activity(see Example 6). Upon removal of the Zn, the cysteines became prone tooxidation and disulfide-linked Apo-2L dimers were formed which haddecreased apoptotic activity. Since the metal binding site appears to beburied in the Apo-2L trimer structure and is not expected to contactreceptor, the data suggests that divalent metal ion binding may beimportant to maintain the trimer structure and stability of Apo-2L.

In order to map Apo-2 ligand's receptor binding site, amino acidresidues important for receptor binding and biological activity wereidentified by alanine-scanning mutagenesis. [Cunningham et al., Science,244:1081-1085 (1989)]. Single alanine substitutions at residues Arg149,Gln205, Val207, Tyr216, Glu236, or Tyr237 resulted in a greater than5-fold decrease in apoptotic activity in a bioassay and showed decreasedaffinity for the receptors (See Examples 3 and 4). Apo-2L binding toDR4, DR5 and DcR2 was most affected by alanine substitutions at residuesGln205, Tyr216, Glu236, or Tyr237, which resulted in at least a 5-folddecreased affinity against all three receptors. All of these variantswith reduced apoptotic activity also exhibited impaired binding toeither DR4 or DR5 (or both) suggesting that receptor binding is requiredfor apoptotic activity.

Alanine substitutions at residues Asp218 and Asp269 resulted in Apo-2Lvariants having increased apoptotic activity. (See Example 4). ResidueAsp218 is located near Tyr216, which is one of the required residues forapoptotic activity. A comparison to the low resolution Apo-2L structure(114-281 form) suggests that the conformation of the 216-220 loop doesnot appear to be significantly altered by the presence of the D218Amutation.

When the results of the mutagenesis analysis were mapped to the Apo-2Ltrimer structure, the functional epitope on Apo-2L for receptor bindingand biological activity was found to be located on the surface formed bythe junction of two monomers (see FIG. 5), similar to TNF-beta. Ashallow groove at the monomer-monomer interface forms the receptorbinding site with both monomers contributing to the binding site.Residues Arg32, Tyr87, and Asp143 in TNF-alpha (corresponding to Apo-2Lresidues Arg158, Tyr216, and Asp267) also make contributions to TNFreceptor binding. [Goh et al., Protein Engineering, 4:785-791 (1991)].In contrast, residues of TNF-alpha (corresponding to residues Gln205,Glu236, and Tyr237 of Apo-2L) play only a minor role in TNFR binding.Thus, while for TNF-alpha the base of the trimer structure makes themost important contribution to receptor binding, in Apo-2L, importantreceptor binding residues are also presented on the top of the trimerstructure. Apo-2L appears to be unique among the TNF family members ofknown structure in having a larger and more extended contact surface forinteraction with its target receptors. It is believed that preferredApo-2L variants will comprise native residues (i.e., will not bemutated) at positions corresponding to Arg149, Gln205, Val207, Tyr216,Glu236, and/or Tyr237.

The description below relates to methods of producing Apo-2 ligand byculturing host cells transformed or transfected with a vector containingApo-2 ligand encoding nucleic acid and recovering the polypeptide fromthe cell culture.

The DNA encoding Apo-2 ligand may be obtained from any cDNA libraryprepared from tissue believed to possess the Apo-2 ligand mRNA and toexpress it at a detectable level. Accordingly, human Apo-2 ligand DNAcan be conveniently obtained from a cDNA library prepared from humantissues, such as the bacteriophage library of human placental cDNA asdescribed in WO97/25428. The Apo-2 ligand-encoding gene may also beobtained from a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the Apo-2ligand or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding Apo-2 ligand is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

Amino acid sequence fragments or variants of Apo-2 ligand can beprepared by introducing appropriate nucleotide changes into the Apo-2ligand DNA, or by synthesis of the desired Apo-2 ligand polypeptide.Such fragments or variants represent insertions, substitutions, and/ordeletions of residues within or at one or both of the ends of theintracellular region, the transmembrane region, or the extracellularregion, or of the amino acid sequence shown for the full-length Apo-2ligand in FIG. 1 (SEQ ID NO:1). Any combination of insertion,substitution, and/or deletion can be made to arrive at the finalconstruct, provided that the final construct possesses, for instance, adesired biological activity or apoptotic activity as defined herein. Ina preferred embodiment, the fragments or variants have at least about80% amino acid sequence identity, more preferably, at least about 90%sequence identity, and even more preferably, at least 95%, 96%, 97%, 98%or 99% sequence identity with the sequences identified herein for theintracellular, transmembrane, or extracellular domains of Apo-2 ligand,or the full-length sequence for Apo-2 ligand. The amino acid changesalso may alter post-translational processes of the Apo-2 ligand, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the Apo-2 ligand sequence as described above can be madeusing any of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Theseinclude oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis.

Scanning amino acid analysis can be employed to identify one or moreamino acids along a contiguous sequence. Among the preferred scanningamino acids are relatively small, neutral amino acids. Such amino acidsinclude alanine, glycine, serine and cysteine. Alanine is typically apreferred scanning amino acid among this group because it eliminates theside-chain beyond the beta-carbon and is less likely to alter themain-chain conformation of the variant. [Cunningham et al., Science,244:1081 (1989)]. Alanine is also typically preferred because it is themost common amino acid. Further, it is frequently found in both buriedand exposed positions [Creighton, The Proteins, (W.H. Freeman & Co.,NY); Chothia, J. Mol. Biol., 150:1 (1976)].

Particular Apo-2L variants of the present invention include those Apo-2Lpolypeptides which include one or more of the recited alaninesubstitutions provided in TABLE I below. Such Apo-2L variants willtypically comprise a non-naturally occurring amino acid sequence whichdiffers from a native Apo-2L amino acid sequence (such as provided inFIG. 1; SEQ ID NO:1, for a full length or mature form of Apo-2L or anextracellular domain sequence thereof) in at least one or more aminoacids. Optionally, the one or more amino acids which differ in theApo-2L variant as compared to a native Apo-2L will comprise amino acidsubstitution(s) such as those indicated in Table I. Apo-2L variants ofthe invention include soluble Apo-2L variants comprising residues91-281, 92-281, 95-281 or 114-281 of FIG. 1 (SEQ ID NO:1) and having oneor more amino acid substitutions recited in TABLE I. Preferred Apo-2Lvariants will include those variants comprising residues 91-281, 92-281,95-281 or 114-281 of FIG. 1 (SEQ ID NO:1) and having one or more aminoacid substitutions recited in TABLE I which enhance biological activity,such as receptor binding.

Variations in the Apo-2 ligand sequence also included within the scopeof the invention relate to amino-terminal derivatives or modified forms.Such Apo-2 ligand sequences include any of the Apo-2 ligand polypeptidesdescribed herein having a methionine or modified methionine (such asformyl methionyl or other blocked methionyl species) at the N-terminusof the polypeptide sequence.

The nucleic acid (e.g., cDNA or genomic DNA) encoding native or variantApo-2 ligand may be inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. Various vectorsare publicly available. The vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence, each of which isdescribed below. Optional signal sequences, origins of replication,marker genes, enhancer elements and transcription terminator sequencesthat may be employed are known in the art and described in furtherdetail in WO97/25428.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the Apo-2ligand nucleic acid sequence. Promoters are untranslated sequenceslocated upstream (5′) to the start codon of a structural gene (generallywithin about 100 to 1000 bp) that control the transcription andtranslation of a particular nucleic acid sequence, such as the Apo-2ligand nucleic acid sequence, to which they are operably linked. Suchpromoters typically fall into two classes, inducible and constitutive.Inducible promoters are promoters that initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g., the presence or absence of a nutrient or achange in temperature. At this time a large number of promotersrecognized by a variety of potential host cells are well known. Thesepromoters are operably linked to Apo-2 ligand encoding DNA by removingthe promoter from the source DNA by restriction enzyme digestion andinserting the isolated promoter sequence into the vector. Both thenative Apo-2 ligand promoter sequence and many heterologous promotersmay be used to direct amplification and/or expression of the Apo-2ligand DNA.

Promoters suitable for use with prokaryotic and eukaryotic hosts areknown in the art, and are described in further detail in WO97/25428.

A preferred method for the production of soluble Apo-2L in E. coliemploys an inducible promoter for the regulation of product expression.The use of a controllable, inducible promoter allows for culture growthto the desirable cell density before induction of product expression andaccumulation of significant amounts of product which may not be welltolerated by the host.

Three inducible promoter systems (T7 polymerase, trp and alkalinephosphatase (AP)) have been evaluated by Applicants for the expressionof Apo-2L (form 114-281). The use of each of these three promotersresulted in significant amounts of soluble, biologically active Apo-2Ltrimer being recovered from the harvested cell paste. The AP promoter ispreferred among these three inducible promoter systems tested because oftighter promoter control and the higher cell density and titers reachedin harvested cell paste.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures can be used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced using standard techniques known in the art.[See, e.g., Messing et al., Nucleic Acids Res., 9:309 (1981); Maxam etal., Methods in Enzymology, 65:499 (1980)].

Expression vectors that provide for the transient expression inmammalian cells of DNA encoding Apo-2 ligand may be employed. Ingeneral, transient expression involves the use of an expression vectorthat is able to replicate efficiently in a host cell, such that the hostcell accumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector [Sambrook et al., supra]. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of Apo-2 ligand that arebiologically active Apo-2 ligand.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of Apo-2 ligand in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes for this purpose include but are not limited to eubacteria,such as Gram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. lichenifonnis (e.g., B. lichenifonnis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. Preferably, the host cell should secreteminimal amounts of proteolytic enzymes.

E. coli is the preferred host cell for use in the present invention. E.coli is particularly well suited for the expression of Apo-2 ligand(form 114-281), a polypeptide of under 20 kd in size with noglycosylation requirement. As a production host, E. coli can be culturedto relatively high cell density and is capable of producing relativelyhigh levels of heterologous proteins.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for Apo-2ligand-encoding vectors. Suitable host cells for the expression ofglycosylated Apo-2 ligand are derived from multicellular organisms.Examples of all such host cells, including CHO cells, are describedfurther in WO97/25428.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for Apo-2 ligandproduction and cultured in nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in Sambrook et al., supra, orelectroporation is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published29 Jun. 1989. In addition, plants may be transfected using ultrasoundtreatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) may be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Prokaryotic cells used to produce Apo-2 ligand may be cultured insuitable culture media as described generally in Sambrook et al., supra.Particular forms of culture media that 25, may be employed for culturingE. coli are described further in the Examples below. Mammalian hostcells used to produce Apo-2 ligand may be cultured in a variety ofculture media.

Examples of commercially available culture media include Ham's F10(Sigma), Minimal Essential Medium (“MEM”, Sigma), RPMI-1640 (Sigma), andDulbedco's Modified Eagle's Medium (“DMEM”, Sigma). Any such media maybe supplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleosides (such as adenosine and thymidine), antibiotics (suchas Gentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRLPress, 1991).

In accordance with the present invention, one or more divalent metalions will typically be added to or included in the culture media forculturing or fermenting the host cells. The divalent metal ions arepreferably present in or added to the culture media at a concentrationlevel sufficient to enhance storage stability, enhance solubility, orassist in forming stable Apo-2L trimers coordinated by one or more zincions. The amount of divalent metal ions which may be added will bedependent, in part, on the host cell density in the culture or potentialhost cell sensitivity to such divalent metal ions. At higher host celldensities in the culture, it may be beneficial to increase theconcentration of divalent metal ions. If the divalent metal ions areadded during or after product expression by the host cells, it may bedesirable to adjust or increase the divalent metal ion concentration asproduct expression by the host cells increases. It is generally believedthat trace levels of divalent metal ions which may be present in typicalcommonly available cell culture media may not be sufficient for stabletrimer formation. Thus, addition of further quantities of divalent metalions, as described herein, is preferred.

The divalent metal ions are preferably added to the culture media at aconcentration which does not adversely or negatively affect host cellgrowth, if the divalent metal ions are being added during the growthphase of the host cells in the culture. In shake flask cultures, it wasobserved that ZnSO₄ added at concentrations of greater than 1 mM canresult in lower host cell density. Those skilled in the art appreciatethat bacterial cells can sequester metal ions effectively by formingmetal ion complexes with cellular matrices. Thus, in the cell cultures,it is preferable to add the selected divalent metal ions to the culturemedia after the growth phase (after the desired host cell density isachieved) or just prior to product expression by the host cells. Toensure that sufficient amounts of divalent metal ions are present,additional divalent metal ions may be added or fed to the cell culturemedia during the product expression phase.

The divalent metal ion concentration in the culture media should notexceed the concentration which may be detrimental or toxic to the hostcells. In the methods of the invention employing the host cell, E. coli,it is preferred that the concentration of the divalent metal ionconcentration in the culture media does not exceed about 1 mM(preferably, ≦1 mM). Even more preferably, the divalent metal ionconcentration in the culture media is about 50 micromolar to about 250micromolar. Most preferably, the divalent metal ion used in such methodsis zinc sulfate. It is desirable to add the divalent metal ions to thecell culture in an amount wherein the metal ions and Apo-2 ligand trimercan be present at a one to one molar ratio.

The divalent metal ions can be added to the cell culture in anyacceptable form. For instance, a solution of the metal ion can be madeusing water, and the divalent metal ion solution can then be added orfed to the culture media.

In one embodiment of the invention, the selected Apo-2L (form 114-281)is expressed in E. coli, and during the culturing or fermentation of thecell culture, the process parameters are set such that cellularactivities are conducted at oxygen uptake rates of approximately 1.0 to3.0 mmoles/L-min for cultures at approximately 40-50 gm/L dry cellweight. It is preferred that the newly synthesized nascent Apo-2Lpolypeptides have sufficient time for proper protein folding andtrimerization of Apo-2L monomers. The growth phase of the fermentationprocess is preferably conducted at 30° C. Just prior to the commencementof product expression; the process temperature control set-point mayremain at 30° C. or be down-shifted to 25° C. for the rest of thefermentation. Optionally, it may be desired to increase cell density inthe cell culture, and the above-mentioned parameters may be adjusted (orincreased) accordingly. For instance, it may be advantageous to increasecell density in the cell culture to increase volumetric yield. Oneskilled in the art can, by using routine techniques known in the art,incrementally increase the cell density and incrementally increase theabove-mentioned parameters, if desired.

Expression of the Apo-2L may be measured in a sample directly, forexample, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci.USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, and particularly ³²P. However, other techniques may alsobe employed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionucleotides, fluorescers or enzymes. Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn may be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected. Gene expression, alternatively, maybe measured by immunological methods, such as immunohistochemicalstaining of cells or tissue sections and assay of cell culture or bodyfluids, to quantitate directly the expression of gene product. Withimmunohistochemical staining techniques, a cell sample is prepared,typically by dehydration and fixation, followed by reaction with labeledantibodies specific for the gene product coupled, where the labels areusually visually detectable, such as enzymatic labels, fluorescentlabels, luminescent labels, and the like.

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native Apo-2 ligand polypeptide or against a synthetic peptidebased on the DNA sequences provided herein or against exogenous sequencefused to Apo-2 ligand DNA and encoding a specific antibody epitope.

Apo-2 ligand preferably is recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysates when directly produced without a secretory signal. If the Apo-2ligand is membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or its extracellularregion may be released by enzymatic cleavage.

When Apo-2 ligand is produced in a recombinant cell other than one ofhuman origin, the Apo-2 ligand is free of proteins or polypeptides ofhuman origin. However, it is usually necessary to recover or purifyApo-2 ligand from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogeneous as to Apo-2 ligand. As afirst step, the culture medium or lysate may be centrifuged to removeparticulate cell debris. Apo-2 ligand thereafter is purified fromcontaminant soluble proteins and polypeptides, with the followingprocedures being exemplary of suitable purification procedures: byfractionation on an ion-exchange column; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE or CM; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; gel filtration using, for example, Sephadex G-75;diafiltration and protein A Sepharose columns to remove contaminantssuch as IgG.

In a preferred embodiment, the Apo-2 ligand can be isolated by affinitychromatography. Apo-2 ligand fragments or variants in which residueshave been deleted, inserted, or substituted are recovered in the samefashion as native Apo-2 ligand, taking account of any substantialchanges in properties occasioned by the variation. For example,preparation of an Apo-2 ligand fusion with another protein orpolypeptide, e.g., a bacterial or viral antigen, facilitatespurification; an immunoaffinity column containing antibody to theantigen can be used to adsorb the fusion polypeptide.

A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native Apo-2 ligand may require modification toaccount for changes in the character of Apo-2 ligand or its variantsupon expression in recombinant cell culture.

During any such purification steps, it may be desirable to expose therecovered Apo-2L to a divalent metal ion-containing solution or topurification material (such as a chromatography medium or support)containing one or more divalent metal ions. In a preferred embodiment,the divalent metal ions and/or reducing agent is used during recovery orpurification of the Apo-2L. Optionally, both divalent metal ions andreducing agent, such as DTT or BME, may be used during recovery orpurification of the Apo-2L. It is believed that use of divalent metalions during recovery or purification will provide for stability ofApo-2L trimer or preserve Apo-2L trimer formed during the cell culturingstep.

A preferred method of recovering and purifying the expressed Apo-2L fromprokaryotic host cells (most preferably from bacterial host cells)comprises the following steps: (a) extracting Apo-2L (intracellular)from E. coli cells; (b) stabilizing the properly folded Apo-2L in abuffer solution comprising divalent metal ions and/or reducing agent;(c) purifying the Apo-2L by chromatography using, sequentially, acationic exchanger, a hydroxyapatite and a hydrophobic interactionchromatograph, and (d) selectively eluting Apo-2L in a buffer solutioncomprising divalent metal ions and/or reducing agent from each suchchromatographic support. The divalent metal ions and the reducing agentutilized in such methods may include a Zn sulfate, Zn chloride, Cosulfate, DTT and BME, and more preferably, a Zn sulfate or DTT. An evenmore detailed description of this recovery and purification process isprovided in Example 8 below.

As discussed above, such methods of the invention are applicable anduseful for various other proteins, besides Apo-2L, which have improvedactivity when in a trimerized form or which require trimerization of theprotein for activity.

Formulations comprising Apo-2 ligand and one or more divalent metal ionsare also provided by the present invention. It is believed that suchformulations will be particularly suitable for storage (and maintainApo-2L trimerization), as well as for therapeutic administration.Preferred formulations will comprise Apo-2L and zinc or cobalt. Morepreferably, the formulation will comprise an Apo-2L and zinc or cobaltsolution in which the metal is at a <2× molar ratio to the protein. Ifan aqueous suspension is desired, the divalent metal ion in theformulation may be at a >2× molar ratio to the protein. Using zincsulfate, Applicants have found Apo-2L (form 114-281) precipitates andforms an aqueous suspension at about a 100 mM concentration of zincsulfate in the formulation. Those skilled in the art will appreciatethat at a >2× molar ratio, there may be an upper range of concentrationof the divalent metal ion in the formulation at which the metal canbecome deleterious to the formulation or would be undesirable as atherapeutic formulation.

The formulations may be prepared by known techniques. For instance, theApo-2L formulation may be prepared by buffer exchange on a gelfiltration column.

Typically, an appropriate amount of a pharmaceutically-acceptable saltis used in the formulation to render the formulation isotonic. Examplesof pharmaceutically-acceptable carriers include saline, Ringer'ssolution and dextrose solution. The pH of the formulation is preferablyfrom about 6 to about 9, and more preferably from about 7 to about 7.5.Preferably, the pH is selected so as to ensure that the zinc remainsbound to the Apo-2L. If the pH is too high or too low, the zinc does notremain bound to the Apo-2L and as a result, dimers of Apo-2L will tendto form. It will be apparent to those persons skilled in the art thatcertain carriers may be more preferable depending upon, for instance,the route of administration and concentrations of Apo-2 ligand anddivalent metal ions.

Therapeutic compositions of the Apo-2L can be prepared by mixing thedesired Apo-2L molecule having the appropriate degree of purity withoptional pharmaceutically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, 16th edition, Oslo, A.ed. (1980)), in the form of lyophilized formulations, aqueous solutionsor aqueous suspensions.

Acceptable carriers, excipients, or stabilizers are preferably nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as Tris, HEPES, PIPES, phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; and/ornon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG).

Additional examples of such carriers include ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as glycine, sorbic acid, potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts, or electrolytes such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, andcellulose-based substances. Carriers for topical or gel-based formsinclude polysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylate,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. For all administrations, conventional depot formsare suitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations.

Effective dosages of Apo-2 ligand in the formulations may be determinedempirically, and making such determinations is within the skill in theart. It is presently believed that an effective dosage or amount ofApo-2 ligand may range from about 1 microgram/kg to about 100 mg/kg ofbody weight or more per day. Interspecies scaling of dosages can beperformed in a manner known in the art, e.g., as disclosed in Mordentiet al., Pharmaceut. Res., 8:1351 (1991). Those skilled in the art willunderstand that the dosage of Apo-2 ligand that must be administeredwill vary depending on, for example, the mammal which will receive theApo-2 ligand, the route of administration, and other drugs or therapiesbeing administered to the mammal.

Apo-2L to be used for in vivo administration should be sterile. This isreadily accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. Apo-2Lordinarily will be stored in lyophilized form or in solution ifadministered systemically. If in lyophilized form, Apo-2L is typicallyformulated in combination with other ingredients for reconstitution withan appropriate diluent at the time for use. An example of a liquidformulation of Apo-2L is a sterile, clear, colorless unpreservedsolution filled in a single-dose vial for subcutaneous injection.

Therapeutic Apo-2L formulations generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.The formulations are preferably administered as repeated intravenous(i.v.), subcutaneous (s.c.), intramuscular (i.m.) injections orinfusions, or as aerosol formulations suitable for intranasal orintrapulmonary delivery (for intrapulmonary delivery see, e.g., EP257,956).

Apo-2L can also be administered in the form of sustained-releasepreparations. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe protein, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981)and Langer, Chem. Tech., 12: 98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

The Apo-2L and its formulations described herein can be employed in avariety of therapeutic and non-therapeutic applications. Among theseapplications are methods of treating various cancers (provided above)and viral conditions. Such therapeutic and non-therapeutic applicationsare described, for instance, in WO97/25428 and WO97/01633.

An article of manufacture such as a kit containing Apo-2L useful for thediagnosis or treatment of the disorders described herein comprises atleast a container and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds an Apo-2L formulation that is effective for diagnosing or treatingthe condition and may have a sterile access port (for example, thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). The label on, orassociated with, the container indicates that the formulation is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. The article of manufacture may also comprisea second or third container with another active agent as describedabove.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Expression and Purification of Apo-2L Variants

Alanine substitution variants of Apo-2L were constructed byoligonucleotide-directed mutagenesis (Kunkel et al., Proc. Natl. Acad.Sci., 82:488-492 (1985); Kunkel, Methods in Enzymology, 154:367-382(1987)) of a plasmid (pAPOK5) (see FIG. 13), designed for theintracellular E. coli expression of the 91-281 amino acid form of Apo-2Lunder control of the trp promoter. pAPOK5 was constructed by using PCRto clone the Apo-2L cDNA (encoding residues 91-281) into plasmid pS1162which carries the trp promoter. E. coli strain 294 transformed with themutated plasmids were grown to mid-log phase at 37° C. in 250 mL M9media plus 100 μM ZnSO₄, expression was induced by addition of 25 μg/mLbeta-indole acrylic acid, and the cultures were grown overnight at 30°C. Cells were harvested by centrifugation and frozen.

The cell pellet was homogenized in 6 volumes 0.1 M Tris-HCl pH 8, 0.2 MNaCl, 5 mM DTT, 1 mM EDTA, and Apo-2L was isolated from the solublefraction by IMAC on a chelating hiTRAP column (Pharmacia) charged withnickel. The Apo-2L had a weak affinity for immobilized metal and couldbe eluted with low concentrations of imidazole. A final purification wasobtained by cation exchange chromatography on a SP hiTRAP column(Pharmacia). Concentrations of purified Apo-2L variants were determinedby absorbance measurements using an e₂₈₀ of 1.4 mg⁻¹ ml cm⁻¹.

The Apo-2L variants identified by the oligonucleotide-directedmutagenesis are listed in table I.

TABLE I Receptor binding and apoptotic activity of Apo2L variants^(a)Ratio (variant/wild-type) DR4-IgG DR5-IgG DcR2-IgG Apoptosis VariantK_(D) K_(D) K_(D) ED₅₀ Δzinc 6.3 6.6 11.2 90.0 R130A 3 2.7 1.3 1.9 N134A1.0 0.8 1.0 1.5 L136A 3.3 1.5 1.4 0.8 S138A 2.1 1.3 2.2 1.2 N140A 1.41.9 0.9 1.1 S141A 2.3 1.3 2.4 1.3 K142A 2.6 1.9 2.7 2.0 N143A 2.1 2.01.3 1.5 R149A 1.8 2.2 1.6 3.5 S153A 2.3 1.2 2.1 0.9 E155A 1.6 2 1.4 2.5R158A 2.4 1.3 6.5 1.4 S159A 4.7 2.2 3.4 0.9 R170A 1.1 2.2 0.6 0.9 K179A0.9 0.9 1.1 2.0 R191A 7.8 3.9 3.2 2.2 Q193A 1.7 1.1 1.2 2.2 E195A 4.61.4 2.6 0.8 K197D 2 2.1 2.9 1.1 K201A 4.3 2.7 10 2.5 N202A 2.5 2.5 1.93.2 D203A 1.5 1.1 0.6 0.5 Q205A 13.1 6.3 10.8 690 V207A 2.2 2.8 2.1 5.6Y213A 1.3 1 1.5 1.2 Y216A 14.5 8.9 9.0 320 D218A 1.3 1.9 1.1 0.3 C230S4.1 7.1 6.7 8.0 E236A 6.0 9.8 8.4 10.8 Y237A 7.3 5.0 48 8.3 Y240A 1.80.8 1.8 1.1 K251A 1.9 2 2.4 0.8 S259A 4.3 2.0 1.4 3.3 H264A 1.9 2.0 1.43.1 D267A 5.7 1.9 5.5 1.11 D269A 1.7 0.5 0.9 0.2 ^(a)Values shownrepresent the ratio of variant to wild-type. For wild-type Apo-2L(residues 91-281), the Kd values for DR4-IgG, DR5-IgG and DcR2-IgG are0.8 ± 0.3 nM, 0.9 ± 0.4 nM, and 0.3 ± 0.2 nM. Wild-type Apo-2L (residues91-281) gave an ED₅₀ of 24 ± 3.1 ng/mL in the apoptosis assay while the114-281 form of Apo-2L was slightly more active and gave an ED50 of 16.0± 3.6 ng/ml. Only 2-fold changes from wild-type values are considered tobe significant.

Example 2 Crystallography Analysis of Apo-2L

Crystals of Apo-2L (amino acid residues 114-281) were grown in 70 uLsitting drops containing 40 uL protein (at 2.6 mg/mL in 20 mM Tris,pH8.0), 20 uL 50 mM Tris pH 8.0, and 10 uL 8% peg 2K MME over a wellsolution of 50% peg 2K MME at 20° C. and were members of the spacegroupP63 with two monomer in the asymmetric unit and unit cell constantsa=72.5, c=140 Angstrom and diffract to 3.9 Angstrom at room temperature.Crystals of D218A variant (see Example 1) grew in 14 uL sitting dropscontaining 4 uL of 4% MPD and 10 uL protein (1.7 mg/ml in 20 mM Tris pH7.5) over a well solution of 32% MPD at 4° C. and were members of thespacegroup R32 with one monomer per asymmetric unit and unit cellparameters 66.4, c=197.7 Angstrom and diffracted to 1.3 Angstrom at−180° C. with synchroton radiation. Data sets diffracting to 3.9Angstrom for the Apo-2L (residues 114-281) crystals and 1.9 Angstrom forthe D218A variant were measured on a Rigaku rotating anode x-raygenerator equipped with a MAR detector and processed withDENZO/SCALEPACK [Otwinowski et al., Proceedings of the CCP4 StudyWeekend: Data Collection and Processing (eds. Sawyer et al.) pp. 56-62Daresbury Laboratory, Daresbury, England, 1993]. A 1.3 Angstrom data setfor the D218A variant was measured at the Advanced Photon Source atArgonne National Labs and was processed wtih HKL2000/SCALEPACK and had aRsym of 6.4% (34% in the 1.35-1.30 shell), with 100% completeness and aredundancy of 12-fold, and I/<I>=12.4.

The native Apo-2L structure was solved by molecular placement using amodel based on TNF-alpha (pdb code 1TNF) with the program Amore [ActaCryst., D50:760-763 (1994)] and was refined [Brunger, X-PLOR: version3.1, Yale Press, New Haven 1987] with strict 2-fold non-crystallographicrestraints until a R_(free) of 35%. This structure refined against the1.9 Angstrom dataset until a R_(free) of 25% and finally was refinedagainst 1.3 Angstrom data with Refmac and SHELXL [Sheldrick et al.,Methods in Enzymology, pp. 319-343, Academic Press, San Diego 1997] ofR_(free)=22% and R_(factor) of 20% with good geometry (rmsd bonds 0.011Angstrom, rmsd angle 1.7°). All residues fall within the allowed regionsof a Ramachandran plot. During refinement, a 28 sigma peak of electrondensity was observed between symmetry related Cys230 on the trimer axis.This density was modeled as a zinc ion and refined with B-factor of 10.It is believed that a chlorine molecule on the trimer axis is present asthe fourth ligand to the zinc. The final model consists of residues120-130, 142-194, 203-281 with 170 solvent molecules and one zinc ionand one chloride ion. Residues 91-119, 131-141, and 195-202 aredisordered. N-terminal sequencing of several crystals confirmed that theN-terminus is intact while mass spectrometry of the starting materialshows that it is full length.

A summary of the crystallographic data is provided in FIG. 2C.

Example 3 Determination of Receptor Binding Affinity of Apo-2L Variants

Dissociation constants (Kd) for binding of Apo-2L variants (see Table I)to immobilized receptor immunoadhesins were determined from surfaceplasmon resonance (SPR) measurements on a Pharmacia BIAcore 1000.

DR5-IgG (also referred to as Apo-2-IgG) and DcR2-IgG receptorimmunoadhesins were prepared as described in WO98/51793 published Nov.19, 1998 and WO99/10484 published Mar. 9, 1999, respectively. DR4-IgGwas prepared as follows. A mature DR4 ECD sequence (amino acids 1-218;Pan et al., supra) was cloned into a pCMV-1 Flag vector (Kodak)downstream of the Flag signal sequence and fused to the CH1, hinge andFc region of human immunoglobulin G₁ heavy chain as described previously[Aruffo et al., Cell, 61:1303-1313 (1990)]. The immunoadhesin wasexpressed by transient transfection into human 293 cells and purifiedfrom the cell supernatants by protein A affinity chromatography, asdescribed by Ashkenazi et al., Proc. Natl. Acad. Sci., 88:10535-10539(1991)].

The receptor immunoadhesin proteins were coupled to the sensor chipsurface at a level of 1000-2000 resonance units using amine couplingchemistry (Pharmacia Biosensor). Sensorgrams were recorded for Apo-2Lbinding at concentrations ranging from 15.6 nM to 500 nM in 2-foldincrements. The kinetics constants were determined by non-linearregression analysis and used to calculate the binding constants.

The results are shown in Table I.

Example 4 Apoptotic Activity of Apo-2L Variants In Vitro

A bioassay which measures cell viability from the metabolic conversionof a fluorescent dye was used to determine the apoptotic activity ofApo-2L variants. Serial 2-fold dilutions of Apo-2L (form 114-281) orApo-2L variants (see Table I) were made in RPMI-1640 media (Gibco)containing 0.1% BSA, and 50 μL of each dilution was transferred toindividual wells of 96-well Falcon tissue culture microplates. 50 μL ofSK-MES-1 human lung carcinoma cells (ATCC HTB58) (in RMPI-1640, 0.1%BSA) were added at a density of 2×10⁴ cells/well. These mixtures wereincubated at 37° C. for 24 hours. At 20 hours, 25 μL of alamar Blue(AccuMed, Inc., Westlake, Ohio) was added. Cell number was determined bymeasuring the relative fluorescence at. 590 nm upon excitation at 530nm. These data were analyzed by using a 4 parameter fit to calculateED₅₀, the concentration of Apo-2L giving a 50% reduction in cellviability.

Single alanine substitutions at residues Arg149, Gln205, Val207, Tyr216,Glu236 or Tyr237 resulted in a greater than 5-fold decrease in apoptoticactivity in the bioassay and showed decreased affinity for DR4, DR5 andDcR2 (Table I). Apo-2L binding to these receptors was most affected byalanine substitution of Gln205, Tyr216, Glu236 and Tyr237, all of whichresulted in at least a 5-fold decreased affinity for all threereceptors. All of the Apo-2L variants with reduced apoptotic activityalso exhibited impaired binding to either DR4 or DR5 (or both)suggesting that receptor-binding is required for the biological effect.Alanine substitution of Asp218 or Asp269 resulted in a greater than2-fold increase in apoptotic activity. It is noteworthy that mostalanine substitutions have similar effects on both DR4 and DR5 binding,the only exceptions being mutation of Gln193, Glu195, Ser259, His264,and Asp267, all of which had a greater than 5-fold effect on DR4 binding(decreasing affinity) but only a small or negligible effect on DR5binding. Changes in DcR2-binding tended to parallel the effects observedfor DR4-binding. Diminished apoptotic activity appears to be mostclosely linked with decreased DR5-binding suggesting that DR5 isrequired for death signaling in SK-MES in response to Apo-2Ladministration.

Example 5 Elemental and Quantitative Analysis to Determine Metal Contentof Apo-2L

Elemental analysis of Apo-2L was performed by using inductively coupledplasma atomic emission spectrometry (ICP-AES). For this determination, a2 mg/mL solution of Apo-2L (residues 114-281 produced in E. coli usingmethods disclosed in WO97/25428; additional quantities of divalent metalions were not added during fermentation or purification in accordancewith the methods described herein) formulated in 20 mM Tris pH 7.5 wasused. Levels of Cd, Co, Zn, Ni, and Cu in this sample and in a portionof the formulation buffer were determined.

TABLE II Sample Cd Co Zn Ni Cu Buffer −0.058 −0.090 −0.098 −0.098 −0.082Apo-2L −0.058   0.199   1.712 −0.108 −0.075

The metals bound to the Apo-2L were Zn and Co (Table II). The calculatedmolar ratios were 0.79 moles of Zn per mole of Apo-2L trimer and 0.06moles of Co per mole of Apo-2L trimer. These data indicate Apo-2L hasone zinc binding site per trimer. The site is 85% occupied with metal inthis preparation of Apo-2L.

Example 6 Effects of Removal of Bound Zinc from Apo-2L Using ChelatingAgents

A sample of Apo-2L (form 114-281) was treated with chelating agents toremove the bound zinc. The sample was first dialyzed against 2 changesof 1000 volumes each of 50 mM EDTA, then against 2 changes of 1000volumes of 2 mM 1,10-phenanthroline, and finally against 1000 volumes ofmetal-free 20 mM Tris pH 7.5. The sample, before and after the chelatingtreatment, was assayed for receptor-binding, metal content and apoptoticactivity. Receptor binding was measured as described in Example 3, metalcontent was determined by ICP-AES and apoptotic activity was assayed asdescribed in Example 4. ICP-AES showed that the dialysis procedureremoved the bound zinc. After this treatment, the receptor affinity wassignificantly reduced (Table I) and the apoptotic activity was decreased90-fold (FIG. 4).

Circular dichroic spectra were recorded on an AVIV instrument (Lakewood,N.J.) model 202 spectropolarimeter. The spectrum was scanned from 250 to200 nm using a step size of 0.5 nm, an averaging time of 5 seconds, andquartz rectangular cuvettes having a pathlength of 1 cm. The proteinconcentration was 2 μM in solutions containing PBS. As shown in FIG. 6,Apo-2L (form 114-281) gives a CD spectrum typical of a protein having ahigh beta-sheet content. Removal of the bound zinc results in adecreased intensity of the dichroic peaks suggesting that the beta-sheetcontent has been diminished.

Circular dichroism was used to monitor the effect of zinc removal on thethermal stability of Apo-2L (form 114-281). These experiments also used1 cm quartz rectangular cuvettes and a protein concentration of 2 μM.Circular dichroism at 225 nm was monitored as the sample was heated from30 to 100° C. Measurements were taken at 2° C. increments after allowingthe sample to equilibrate at that temperature for 1 minute. Both theellipticity (CD) and dynode voltage were recorded and the temperaturedependence of the dynode voltage is plotted in FIG. 7. The dynodevoltage is proportional to the absorbance of the sample. An increase indynode voltage upon heating of the sample reflects protein aggregation.The increase in dynode voltage was concomitant with a loss of secondarystructure as indicated by a decrease in the ellipticity at 225 nm. Thesedata suggest that Apo-2L (form 114-281) aggregates upon thermaldenaturation with the midpoint for this transition (Tm) occurring atabout 75° C. Removal of the bound zinc results in a large decrease inthe Tm for thermal denaturation to about 54° C. These data show that thebound zinc is necessary for maintenance of the structure and stabilityof homotrimeric Apo-2L.

Example 7

Effects of Removal of Zinc Binding Site from Apo-2L by Mutation

Cys230 of Apo-2L (form 91-281) was replaced with Ala or Ser by usingoligonucleotide-directed mutagenesis as described in Example 1. Thevariant proteins were then expressed and purified as described inExample 1. As shown in Table I, both the C230A and C230S mutants ofApo-2L (form 91-281) had reduced receptor-binding affinity and greatlydiminished apoptotic activity. Since the Apo-2L x-ray structure showsthat Cys230 is a buried residue, and thus is unlikely to directlycontact receptor upon complex formation, these data suggest thatmutation of Cys230 indirectly affects activity by changes in thestructure or stability of homotrimeric Apo-2L.

Example 8

Additions of Zn Improves Soluble Apo-2 Ligand Product Accumulation andRecovery

A. Apo-2L (Amino Acid Residues 114-281) Expression Regulated by theAlkaline Phosphatase Promoter

pAPApo2-P2RU (see FIG. 12) encodes for the co-expression of Apo-2L(amino acid residues 114-281) and the tRNA's encoded by pro2 and argU.The pBR322-based plasmid [Sutcliffe, Cold Spring Harbor Symp. Quant.Biol., 43:77-90 (1978)] pAPApo2-P2RU was used to produce the Apo-2L inE. coli. The transcriptional and translational sequences required forthe expression of Apo-2L are provided by the alkaline phosphatasepromoter and the trp Shine-Dalgarno, as described for the plasmid phGH1[Chang et al., Gene, 55:189-196 (1987)]. The coding sequence for Apo-2L(form 114-281) is located downstream of the promoter and Shine-Dalgarnosequences and is preceded by an initiation methionine. The codingsequence includes nucleotides (shown in FIG. 1) encoding residues114-281 of Apo-2L (FIG. 1) except that the codon encoding residue Pro119is changed to “CCG” instead of “CCT” in order to eliminate potentialsecondary structure. The sequence encoding the lambda to transcriptionalterminator [Scholtissek et al., Nucleic Acids Res., 15:3185 (19.87)]follows the Apo-2L coding sequence. Additionally, this plasmid alsoincludes sequences for the expression of the tRNA's pro2 [Komine et al.,J. Mol. Biol., 212:579-598 (1990)] and argU/dnaY [Garcia et al., Cell,45:453-459 (1986)]. These genes were cloned by PCR from E. coli w3110and placed downstream of the lambda to transcriptional terminatorsequence. This plasmid confers both tetracycline and ampicillinresistance upon the production host.

Strain 43E7 (E. coli W3110 fhuA(tonA) phoA Δ(argF-lac) ptr3 degP kanSompT ilvG+) was used as the production host for the co-expression of theApo-2 ligand and the tRNA's. Competent cells of 43E7 were prepared andtransformed with pAPApo2-P2RU using standard procedures. Transformantswere picked from LB plates containing 20 μg/ml tetracycline (LB+Tet20),streak-purified, and grown in LB broth with 20 μg/ml tetracycline in a30° C. shaker/incubator before being stored in DMSO at −80° C.

A shake flask inoculum was prepared by inoculating sterile medium usinga freshly thawed stock culture vial. Appropriate antibiotics wereincluded in the medium to provide selective pressure to ensure retentionof the plasmid. The shake flask medium composition is given in TableIII. Flask cultures were incubated with shaking at about 30° C. (28°C.-32° C.) for 14-18 hours. This culture was then used to inoculate theproduction fermentation vessel. The inoculation volume was between 0.1%and 10% of the initial volume of medium.

TABLE III Shake Flask Medium Composition Ingredient Quantity/LiterTetracycline 4-20 mg Tryptone 8-12 g Yeast extract 4-6 g Sodium chloride8-12 g Sodium phosphate, added as pH7 4-6 mmol solution

Production of the Apo-2L was carried out in the production medium givenin Table IV. The fermentation process was conducted at about 30° C.(28-32° C.) and pH controlled at approximately 7.0 (6.5-7.5). Theaeration rate and the agitation rate were set to provide adequatetransfer of oxygen to the culture. At the onset of product expression,induced by phosphate depletion, the process temperature was shifted from30° C. to 25° C. Throughout the fermentation process, the cell culturewas fed glucose based on a computer algorithm to meet its carbonrequirement while ensuring aerobic condition.

Two batch additions of ZnSO₄ were made during the fermentation process.One addition was made just prior to the induction of product expression.The second addition was made at approximately the mid-point of theproduction period. In this example, the additions occurred at a cultureoptical density of about 80-120 OD₅₅₀ and at about 28 hourspost-inoculation. Sufficient amounts of 100 mM ZnSO₄ were added toachieve approximately 50-100 micromolar (final concentration) with eachbatch addition of the metal ions.

The fermentation was allowed to proceed for about 34-45 hours, afterwhich the cell paste was harvested from the broth for subsequent productrecovery evaluation.

TABLE IV Production Medium Composition for AP Promoter Expression SystemIngredient Quantity/Liter Tetracycline 4-20 mg Glucose^(a) 10-250 gAmmonium sulfate^(a) 2-8 g Sodium phosphate, monobasic, 1-5 gdihydrate^(a) Potassium phosphate, dibasic^(a) 1-5 g Potassiumphosphate, monobasic^(a) 0-5 g Sodium citrate, dihydrate^(a) 0.5-5 gPotassium chloride 0-5 g Magnesium sulfate, heptahydrate^(a) 1.0-10 gAntifoam 0-5 ml Ferric chloride, hexahydrate^(a) 20-200 mg Zinc sulfate,heptahydrate^(a) 0.2-20 mg Cobalt chloride, hexahydrate^(a) 0.2-20 mgSodium molybdate, dihydrate^(a) 0.2-20 mg Cupric sulfate,pentahydrate^(a) 0.2-20 mg Boric acid^(a) 0.2-20 mg Manganese sulfate,monohydrate^(a) 0.2-20 mg Casein hydrolysate^(a) 5-25 g Yeastextract^(a) 5-25 g ^(a)A portion of these ingredients may be fed to theculture during the fermentation. Ammonium hydroxide was added asrequired to control pH.

Broth samples were taken over the time course of the fermentationprocess. Cells from 1 ml of broth samples diluted to a cell density of20 OD₅₅₀ were collected by centrifugation and the resultant cell pelletswere stored at −20° C. until analysis. The cell pellets were thawed andresuspended in 0.5 ml of extraction buffer (50 mM HEPES, pH 8.0, 50 mMEDTA and 0.2 mg/ml hen egg-white lysozyme) and mechanically disrupted torelease the product from the cytoplasm. Solids were removed from thecell lysates by centrifugation before the clarified lysates were loadedonto a Dionex ProPac IEX HPLC column for trimer quantitation. The HPLCassay method resolves the product away from the contaminating E. coliproteins by use of a 5%-22% gradient of 1M NaCl in a 25 mM phosphate (pH7.5) buffer over 25 minutes at a flow rate of 0.5 ml/min.

Frozen cell paste was thawed and resuspended in extraction buffer (100mM Hepes buffer, pH 8.0, 50 mM EDTA, 5 mM DTT). After multiple passes ofthe cell suspension through a mechanical homogenizer to release theApo-2L product from the cytoplasmic compartment, 0.2% PEI (finalconcentration) was added and the solids were removed by centrifugation.The clarified lysate was diluted 1:1 (v/v) with H₂0 and pH adjusted to7.2 prior to loading onto the MPHS column (BioRad) pre-equilibrated with3-4 column volumes of 50 mM Hepes/0.05% Triton/1 mM DTT pH 7.2. After awash step with the 2-3 column volumes of equilibration buffer followedby a second wash step with 0.1M NaCl in the equilibration buffer, Apo-2Lproteins were eluted off the MPHS column with 6 column volumes of a 0.1Mto 0.8M NaCl gradient in the equilibration buffer. Column eluantfractions were collected, analyzed and the relevant fractions werepooled and stored @ 4-8° C.

Analysis of the Apo-2L accumulation during fermentation showed that theZnSO₄ additions did not significantly affect cell growth. The productionof Apo-2 ligand began when the phosphate in the medium was depleted,typically about 15-25 hours after inoculation. FIG. 8 shows the timecourse in the accumulation of soluble Apo-2L trimers detected by the IEXHPLC method. Cultures with ZnSO₄ additions had higher productconcentration in the cell lysate samples than the minus-ZnSO₄ controls.

Analysis of the product recovery at the initial capture step (involvingIEX Chromatography) (FIG. 9) shows the elution profiles of the MPHSchromatography of cell lysates from fermentations conducted in theabsence and presence of ZnSO₄ additions. After the initial flow-throughand wash steps, two main peaks, Peak A and Peak B, were resolved. BySDS-PAGE analysis, both peaks consisted of mainly Apo-2L.

Purified material from both peaks was prepared and analyzed forbiological activity and stability. Results obtained from these studiessuggested that Peak A was a more stable pool of Apo-2L product whilePeak B had a greater tendency to aggregate over time. To minimizeinstability, Peak B was excluded for further recovery. The ratio of PeakA to Peak B was estimated by weighing the cut out traces representingthe integrated area under each of the peaks. Results tabulated in TableV show a shift of the percent of Apo-2L as Peak A from approximately 45%on the average for the minus-ZnSO₄ case to approximately 80% for theplus-ZnSO₄ case, a significant increase in the amount of Apo-2L productin the recoverable pool.

TABLE V ZnSO4 Chromatography Peak Peak Run ID Additions Scale A % B %SAPO2-113 No 0.66 × 15.5 cm 41.6 58.4 LAPO2-4 No  4.4 × 41.5 cm 50.449.6 SAPO2-138 Yes 0.66 × 17.0 cm 78.8 21.2B. Apo-2L (Amino Acid Residues 114-281) Expression Regulated by the trpPromoter:

PS1346.Apo2L.0 Plasmid Construction: DNA encoding residues 114-281 ofApo-2L (preceded by an initiating methionine codon) was inserted into apS1346 plasmid vector. The pS1346 plasmid is a derivative of pHGH207-1[DeBoer et al., Promoters: Structure and Function, Praeger, New York,pp. 462-481 (1982)] and contains the lambda-to transcriptionalterminator [Scholtissek et al., Nucleic Acids Res., 15:3185 (1987)]downstream of the Apo-2L encoding sequence.

Strain 54C2 (E. coli W3110 fhuA(tonA) ion galE rpoHts(htpRts) clpPlacIq) was used as the production host for the expression of Apo-2ligand (amino acid residues 114-281) where the ligand expression wasregulated by the trp promoter. Competent cells of 54C2 were prepared andtransformed with pS1346.Apo2L.0 using standard procedures. Transformantswere picked from LB plates containing 20 μg/ml tetracycline (LB+Tet20),streak purified, and grown in LB broth with 20 μg/ml tetracycline in a30° C. shaker/incubator before being stored in DMSO at −80° C.

Experiments conducted with the production organism, 54C2/pS1346.Apo2L.0,were performed under similar fermentation parameters as described abovefor the AP-promoter expression system except for minor adjustments inthe medium composition (Table VI), the addition of an inducer,indole3-acrylic acid (IAA), and the length of the process. Tryptophanwas added to the initial medium to repress promoter activity during theinitial growth phase. The temperature shift from 30° C. to 25° C. wasmade when cell density of the broth reached approximately 30 OD₅₅₀. Theinducer was added when the cell density of the broth reachedapproximately 55 OD₅₅₀. In the experiments where ZnSO₄ additions weremade, sufficient amounts of 100 mM ZnSO₄ solution were added at celldensity of 25 OD₅₅₀ and at 24 hours post-inoculation to achieve a finalconcentration of approximately 50-100 μM. Cell pastes were harvested at6 hours post inducer addition and stored at −20° C. to −80° C.

TABLE VI Additions to the Production Medium for AP Promoter ExpressionSystem necessary for the trp Promoter Expression System IngredientQuantity/Liter L-isoleucine 0.5-1 g Tryptophan 0.1-5 g

Cell growth for the trp promoter system slowed after reaching 40-60OD₅₅₀. There was significant leaky expression of Apo-2 ligand prior toinduction with IAA addition and was probably responsible for the growthproblem. Cell growth profiles were comparable in the absence and thepresence of ZnSO₄ additions. FIG. 10 shows the time course in theaccumulation of soluble Apo-2L trimers detected by the IEX HPLC method.The accumulation of soluble Apo-2L continued to increase in the run withZnSO₄ additions and achieved a higher product concentration in the celllysate samples.

C. Apo-2L (amino acid residues 114-281) recovery and purification fromE. coli using divalent metal ions/DTT:

The following protocol may be employed in recovery and purification ofApo-2L from E. coli. First, the cells are homogenized and extraction isperformed as follows. Frozen harvested Escherichia coli cells aresuspended in 6 volumes of extraction buffer (100 mM HEPES/5 mM DTT (or 5mM Zn sulfate instead of DTT), pH 8.0), or whole cell broth isconditioned with 5 mM DTT (or 5 mM Zn sulfate instead of DTT) @ pH 8.0.The suspension is thoroughly mixed for 1 hour at 2-8° C., thenhomogenized in a homogenizer (Gaulin Corporation, Everett, Mass.). Thebroken cell suspension is flocculated in 0.2% PEI for 1-2 hours andcentrifuged by a BTPX205 (Alfa Laval Separation AB, Sweden) continuousfeed centrifuge and clarified by depth filtration.

After extraction, the Apo-2L is purified as follows. Macro-Prep ceramicHigh S (MP-HS) chromatography is performed by conditioning the clarifiedcell suspension (extract) with an equal volume of H₂O/0.1% Triton-100and adjusted pH to 7.2. The conditioned extract is loaded onto a columnof MP-HS cation exchanger (Bio-Rad, Hercules, Calif.) that isequilibrated in 50 mM HEPES/0.05% Triton-100/1 mM DTT (or 100 uM Znsulfate), pH 7.2. (In the preceding two steps, SP-Sepharose Fast Flow(Amersham Pharmacia, Sweden) may alternatively be employed). Thenon-binding proteins are flowed through and removed by washing withequilibration buffer to baseline @ A280. The column is washed with 3column volumes of 0.1 M NaCl/equilibration buffer. The Apo-2L is elutedusing a linear, 8 column-volume gradient from 0.1 to 0.8M sodiumchloride in equilibration buffer. Fractions are collected and thosewhich contain properly-folded Apo-2L, as determined by SDS-PAGE orSEC-HPLC assay, are pooled.

The pool of Apo-2L from the MP-HS column is loaded onto a column ofMacro-Prep Hydroxyapatite (Bio-Rad, Hercules, Calif.) equilibrated in 50mM HEPES/1 mM DTT (or 100 uM Zn sulfate), pH 7.2. (As an alternative tothe Macro-Prep Hydroxyapatite, SP-Sepharose Fast Flow may be employed).After the sample is loaded, the column is washed with equilibrationbuffer to baseline @ A280. The Apo-2L is eluted out of the column byusing an isocratic step of 0.15 M sodium phosphate in equilibrationbuffer.

The pool of MP-HA is conditioned with an equal volume of 1.0 M AmmoniumSulfate/50 mM Tris/1 mM DTT (or 100 uM Zn sulfate), pH 7.5, and thenloaded onto a column of Phenyl-Sepharose FF (Amersham Pharmacia, Sweden)that is equilibrated in 0.5 M Ammonium sulfate/50 mM Tris/1 mM DTT (or100 uM Zn sulfate), pH 7.5. (As an alternative to the 0.5M ammoniumsulfate, 0.6M sodium sulfate may be employed). The column is washed withequilibration buffer, and the Apo-2L is collected in the columneffluent.

The Apo-2L is then formulated by ultrafiltration and G-25 Gel Filtration(Amersham Pharmacia, Sweden) chromatography. The pool ofphenyl-Sepharose is concentrated with TFF Ultrafiltration (Millipore,Bedford, Mass.) and formulated on a G-25 gel filtration column with 20mM Tris/8% Trehalose, pH 7.5. (As an alternative, the material may beformulated by diafiltration). The final purity of Apo-2L can bedetermined by SDS-PAGE, SEC-HPLC and Amino Acid sequence analysis.

Example 9 Additions of Cobalt Chloride Improve Soluble Apo-2 LigandProduct Accumulation and Recovery

Apo-2L (Amino Acid Residues 114-281) Expression Regulated by theAlkaline Phosphatase Promoter

The same production organism, medium composition, fermentationconditions and sample analysis described in Example 8A were used instudying the effect of additions of metal ion other than Zn, namelycobalt chloride, on Apo-2L (amino acid residues 114-281) productaccumulation. A solution of 100 mM CoCl₂ in H₂O was used in place of the100 mM ZnSO₄ and sufficient amounts were added to arrive at a finalconcentration of 50-100 μM at each of the two additions.

FIG. 11 shows the benefits of additions of CoCl₂ on soluble Apo-2Laccumulation during the fermentation process. Like the ZnSO₄ additionexperiments, though not as significant, a higher accumulation rate ofsoluble Apo-2L product was detected by the IEX HPLC assay method. Thedata demonstrates that additions of certain metal ions generally improvesoluble Apo-2L accumulation, likely as a result of their ability tostabilize the assembled trimers of Apo-2L.

Example 10 Effects of Various Metal Ions on Apo-2L Formulations

In vitro assays were conducted by incubating 50 μl Apo-2L (form 114-281)at 5° C. for 24 hours with 5 mM of metal salt (each of which are recitedin Table VII below; 100:1 molar ratio of metal to protein) in a 20 mMTris, 8% trehalose, 0.01% Tween 20, pH 7.5 formulation. The samples werethen evaluated for apoptotic activity using a SK-MES assay as describedin Example 4.

The EC50 (or Apo-2L concentration that results in killing of 50% of thecells) is shown in Table VII (units of ng/ml). As shown in Table VII,addition of Zn acetate and Zn sulfate to the culture enhanced Apo-2Lactivity.

TABLE VII Metal Added Bioassay EC50 Control 19.0 ± 1.9 (n = 3)  Mnacetate 19.8 Mn chloride 19.9 Fe acetate 32.5 Co acetate 25.9 Cochloride 17.9 Co sulfate 16.7 Ni acetate 18.7 Cu acetate 844 Cu chloride984 Cu sulfate 774 Ag acetate 20 Zn acetate 12.3 Zn chloride 17.2 Znsulfate (100:1) 9.3 ± 1.2 (n = 3)  (10:1) 10.0 ± 0 (n = 2)    (1:1) 11.5± 0.7 (n = 2) 

Example 11 Effects of Zinc Metal Ions on Apo-2 Ligand FormulationStability

As indicated in Example 8B, the first, step in the purification processusing MPHS cation exchanger gives two peaks, A and B. To investigate thestorage stability of the two peaks, Apo-2L (form 114-281) was extractedfrom the E. coli expressed product (using the pS1346.ApoL.0 plasmid withtrp promoter), and purified by MPHS cation exchanger. The MPHS Peak Awas further purified by MP-Hydroxyapatite and Phenyl-Sepharose FF, andthen formulated using G-25 gel filtration. The MPHS Peak B was purifiedby MP-Hydroxyapatite, Phenyl-Sepharose FF, and Ni-NTA Superflow and thenformulated on G-25 gel filtration into 20 mM Tris, 8% trehalose, 0.01%Tween 20, pH 7.5. The samples were sterile filled into 3 cc glass vialsand sealed with teflon coated greybutyl stoppers. The storage stabilityof the purified and formulated Peaks A and B were evaluated at the time(weeks (“wk”) or months (“mo”)) and temperatures (° C.) listed in TableVIII.

TABLE VIII SDS-SEC SDS-SEC SEC % SEC % RP % % M % D recovery trimer mainEC50 Peak A 1 wk, ND ND 98.60 78.10 −70 C. 1 wk, ND ND 96.7 98.2 73.6037 C. 2 mo, 88.85 7.50 93.93 74.52 −70 C. 2 mo, 84.90 10.58 65.07 94.0353.98 30 C. 6 mo, 93.00 5.80 ND 11.1 −70 C. 6 mo, 87.00 9.44 100 ND 14.85 C. 6 mo, 80.00 8.68 76 ND 67.8 30 C. Peak B 1 wk, ND ND 98.70 75.3 −70C. 1 wk, ND ND 101.1 98.3 61.4 37 C. 2 mo, 94.03 3.79 90.00 65.77 −70 C.2 mo, 94.12 4.54 46.07 94.12 32.185 30 C. 6 mo, 82.00 11.25 ND 10.3 −70C. 6 mo, 73.50 17.00 92 ND 12.6 5 C. 6 mo, 47.00 23.20 38 ND Dead 30 C.

Table VIII shows, among other things, the percent monomer (% M) andpercent dimer (% D); “ND” refers to those values not determined. Peak Aincluded slightly less dimer and other impurities than Peak B at Day 0.As shown in Table VIII, Peak B was found to be more thermally labile, asassessed by the more extensive precipitation at 30° C. (approximately40% more at the 2 month point). Even at 5° C., Peak B was approximately2-fold more prone to dimer formation than Peak A. Biochemical analysisof Peaks A and B indicated that the material of each was biochemicallyequivalent except for their respective stability properties. It isbelieved that the lower stability of Peak B may be related to improper(trimer) assembly or lower zinc content (0.83 molar ratio of Zinc toprotein for Peak A and 0.7 molar ratio of Zinc to protein for Peak B).

1. A method of making Apo-2 ligand, comprising the steps of: (a)providing an E. Coli host cell comprising a replicable vector containinga nucleotide sequence encoding Apo-2 ligand polypeptide; (b) providingculture media containing zinc at a concentration of about 50 micromolarto about 250 micromolar; (c) culturing the host cell in the culturemedia under conditions sufficient to express the Apo-2 ligand; and (d)recovering the Apo-2 ligand from the host cell or culture media, whereinthe Apo-2 ligand comprises a polypeptide selected from the groupconsisting of: (i) a polypeptide having amino acid residues 1 to 281 ofFIG. 1 (SEQ ID NO:1); (ii) a polypeptide having amino acid residues 114to 281 of FIG. 1 (SEQ ID NO:1); (iii) a fragment of the polypeptide of(i) or (ii) which induces apoptosis in at least one type of mammaliancell or binds an Apo-2 ligand receptor; and (iv) a polypeptide having atleast 80% identity to the polypeptide of (i) or (ii), and inducesapoptosis in at least one type of mammalian cell or binds an Apo-2ligand receptor.
 2. The method of claim 1 wherein said zinc compriseszinc sulfate.
 3. The method of claim 1 wherein said replicable vectorcomprises a nucleotide sequence encoding one or more tRNA molecules. 4.The method of claim 3 wherein said replicable vector is the pAPApo2-P2RUvector.
 5. The method of claim 1 wherein said Apo-2 ligand comprisesamino acids 114 to 281 of FIG. 1 (SEQ ID NO:1).
 6. The method of claim 1wherein said Apo-2 ligand comprises amino acids 1 to 281 of FIG. 1 (SEQID NO:1) or a fragment thereof which induces apoptosis in at least onetype of mammalian cell.
 7. The method of claim 1, wherein in step (d),the Apo-2 ligand is recovered from the host cell or culture media in thepresence of a reducing agent.
 8. The method of claim 7, wherein saidreducing agent is selected from the group consisting of DTT and BME.